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. 2021 Oct 9;297(5):101295. doi: 10.1016/j.jbc.2021.101295

Figure 6.

Figure 6

Substitution of the amino acid sequence D36/W37/T38 diminished antagonistic properties of homodimeric p40.A, Western blot analysis of secreted mp40 and mp40D36K/W37K/T38K conditions. In total, 20 μl conditioned supernatant of transiently transfected CHO-K1 was separated by SDS-PAGE under (non-)reducing conditions, and proteins were visualized by Western blotting with a mp40-specific antibody. B, co-IP of FLAG-tagged murine p40 variants (wild-type, D36K/W37K/T38K) and full-length mIL-23R or mIL-12Rβ1. The position of mIL-23R and mIL-12Rβ1 is indicated by arrows. One of two independent experiments is shown. C, co-IP of FLAG-tagged human p40 variants (wild-type, D36K/W37K/Y38K) and full-length hIL-12Rβ1. One of two independent experiments is shown. D, cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 7.5 ng/ml murine HIL-23 with or without 2 μg/ml mp40. The results of one representative experiment of two are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗p ≤ 0.001, ns not significant. E, cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells. The cells were cultured for 3 days in the presence of 5 ng/ml murine HIL-12 with or without 2 μg/ml mp40. The results of one representative experiment of three are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗p ≤ 0.001, ns not significant. F, cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 7.5 ng/ml murine HIL-23 with or without 2 μg/ml mp40D36K/W37K/T38K. The results of one representative experiment of two are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗p ≤ 0.001, ns not significant. G, cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells. The cells were cultured for 3 days in the presence of 5 ng/ml murine HIL-12 with or without 2 μg/ml mp40D36K/W37K/T38K. The results of one representative experiment of three are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗p ≤ 0.001, ∗∗p ≤ 0.01, ns not significant. H and I, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R or Ba/F3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells were washed and starved for at least 4 h. One hour prior to stimulation 2 μg/ml mp40 or mp40D36K/W37K/T38K have been added. Cells were stimulated with the indicated cytokines (7.5 ng/ml HIL-23, 5 ng/ml HIL-12) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of two.